Proanthocyanidins (nonhydrolyzable tannins) are a group of polyphenolic natural products, of current interest because of their numerous biological activities, their widespread occurrence in foodstuffs, and their resulting relevance for human health.
Proanthocyanidins are dimeric or oligomeric flavanoids which have one or several hydroxyl groups on their aromatic rings and often an additional hydroxyl group in the 3 position. Eleven different hydroxylation patterns of the A and B rings have been found in nature. Representative proanthocyanidins include:
 Substitution PatternClassMonomer35783′4′5′Proapi-Apigeni-HOHOHHHOHHgeninidinflavanProluteo-Luteoli-HOHOHHOHOHHlinidinflavanProtri-Triceti-HOHOHHOHOHOHcetinidinflavanPropelar-AfzelechinOHOHOHHHOHHgonidinProcya-CatechinOHOHOHHOHOHHnidinProdel-Gallo-OHOHOHHOHOHOHphinidincatechinProgui-Guibour-OHHOHHHOHHbour-tinidoltinidinProfi-FisetinidolOHHOHHOHOHHsetinidinProrobi-Robine-OHHOHHOHOHOHnetinidintinidolProteraca-OritinOHHOHOHHOHHcinidinPromelac-ProsopinOHHOHOHOHOHHacinidin
The stereochemistry of the substituents on a polyphenol monomeric unit of a proanthocyanidin may be described in terms of their relative stereochemistry, “alpha/beta” or “cis/trans”. The term “alpha” (α) indicates that the substituent is oriented below the plane of the flavan ring, whereas, “beta” (β) indicates that the substituent is oriented above the plane of the ring. The term “cis” indicates that two substituents are oriented on the same face of the ring, whereas “trans” indicates that two substituents are oriented on opposite faces of the ring.
The isolation of pure proanthocyanidins from natural sources becomes increasingly difficult with increasing degree of oligomerization. Degradation by thiolysis permits identification of the underlying monomeric units but the tasks of elucidating the position and stereochemistry of the interflavan linkages is nontrivial. Both of these factors have resulted in few defined oligomers above the tetramer being described in the prior art. Proanthocyanidins and their parent monomers also occur naturally in the form of a variety of derivatives, for example, glycosides or esters with hydroxylated aromatic carboxylic acids, such as gallic or hexahydroxydiphenic acid.
Among the proanthocyanidins, two subtypes, the procyanidins (5,7,3′,4′-hydroxylation) and prodelphinidins (5,7,3′,4′,5′-hydroxylation), are widespread in human foodstuffs, e.g., cocoa. Cocoa procyanidins consist predominantly of epicatechin (the C-3 epimer of catechin) building blocks. Oligomers up to the size of the decamer have been identified. From the pentamer on, these oligomers exhibit growth inhibitory activity against various cancer cell lines. (Romanczk, L. J. Jr.; Hammerstone, J. F., Jr.; Buck, M. M. U.S. Pat. No. 5,554,645, Sep. 10, 1996.) Flavan-3-ols are biosynthetically derived from (2S)-phenylalanine via flavan-3,4-diols. These latter intermediates readily form a highly stabilized carbenium ion (or quinone methide) in position C-4 which attacks the A ring of a flavan-3-ol in what is essentially a Friedel-Crafts alkylation process forming an interfiavan bond. This process can be repeated once or several times, resulting in chain-type oligomers which together with the dimers are known as non-hydrolyzable tannins, condensed tannins, or proanthocyanidins. As one skilled in the art will realize, the structural complexity of these compounds rapidly increases with their chain length as a consequence of different hydroxylation patterns and C-3 stereochemistry in the monomer units and different regio- and stereochemistries of the interflavan linkages, as well as additional structural modifications. In addition, chain branching may occur by alkylation of a monomer unit in both its 6- and 8-positions.
To prove definitively the structures assigned to the compounds purified from cocoa, comparisons must be made to epicatechin dimers and oligomers of defined structure prepared synthetically. Synthetic monomers, dimers and oligomers are useful to develop structure-activity relationships in various in vitro and ultimately in vivo models of anticancer activity.
The synthetic challenge posed by procyanidins is related to the difficulty in controlling the interfiavan regio- and stereochemistry, as well as the sensitivity of the non-protected compounds to acids, bases, and oxidizing agents. The condensation between flavan-3-ols and 4-substituted, electrophilic flavans has traditionally been performed without the use of phenol protecting groups in a mildly acidic medium or recently with AgBF4 for benzylthio as the 4-substituent. The products are mixtures of regio- and sometimes stereoisomers, as well as higher oligomers despite the application of an excess of the nucleophilic building block. They have usually been separated by gel chromatography on Sephadex LH-20, a process that requires a considerable investment of time to develop for each particular separation task because of the unavailability of fast analytical tools such as HPLC columns or thin layer plates for this adsorbent. In addition, optically pure, nonprotected 4-substituted catechins and epicatechins are not readily available, being prepared by reduction of the expensive natural product, (+)-taxifolin (the 4-ketone) or by in situ degradation or thiolytic degradation of natural proanthocyanidin oligomic fractions for which commercial sources are difficult to identify or nonexistant.
It is therefore not surprising that prior art syntheses have used protected oligomeric procyanidins as building blocks. As an additional incentive, protection of the phenolic but not of the alcoholic hydroxyls would permit the regioselective elaboration of derivatives such as 3-esters and -glycosides, as has been done in the case of catechin using acetyl protecting groups. An interesting approach has been reported in which the 8-bromo derivative of 3-O-benzyl-5,7,3′,4′-tetra-O-methylcatechin was subjected to halogen-lithium exchange and reacted with an O-methylated 4-ketone, thus ensuring complete regiocontrol. However, the methyl O-blocking groups cannot be removed to obtain the free dimer. The remaining published work has made use of the above-described electrophilic substitution process with inclusion of phenol protecting groups on one or both of the reaction partners.
Our own previous work directed toward the synthesis of defined epicatechin oligomers used the TiCl4-mediated alkylation of 5,7,3′,4′-tetra-O-benzyl-(−)-epicatechin with 5,7,3′,4′-tetra-O-benzyl-4-(2-hydroxyethoxy)epicatechin. Besides higher oligomers, whose yields rapidly decrease with increased molecular mass, a single dimeric product having beta stereochemistry of the interflavan bond (a procyanidin B2 derivative) was obtained.
Until quite recently, the analytical methods employed for the assignment of interflavan bond regio- and stereochemistry in these compounds were not validated by an independent confirmation of the structure of any dimeric proanthocyanidin. The application of X-ray crystallography has been prevented by the poor crystallizability of proanthocyanidins and their derivatives. Assignments of stereochemistry on the basis of 1H NMR coupling constants and circular dichroism disregard the basic fact that the C rings are conformationally flexible. From a conservative point of view, postulates of specific conformations of flexible molecules, regardless of their source (intuitive or computational), cannot be considered a prudent approach to structure elucidation.
Advances in synthetic methodology, which have taken place after the isolation of numerous proanthocyanidins from natural sources, have now enabled one to obtain a definitive proof of the previously conjectured 4β stereochemistry in procyanidin B2. A differentially protected epicatechin dimer, correlated with procyanidin B2 through a common derivative, was subjected to a series of defunctionalization steps and finally degraded to (R)-(−)-2,4-diphenylbutyric acid, isolated as its benzhydryl ester. The sole remaining asymmetric center of this degradation product is directly derived from C-4 of the “top” epicatechin moiety in procyanidin B2, and the sign of the optical rotation of the degradation product, the absolute configuration of which was established by X-ray crystallography, thus revealed the absolute configuration at C-4.
Those skilled in the art will recognize the importance of having an authentic sample of the opposite stereoisomer, now recognizable by default as epicatechin-4α,8-epicatechin for comparison. Literature reports of proanthocyanidins for which simultaneously a 2,3-cis and a 3,4-cis relationship of their C-ring substituents has been postulated are scarce, and epicatechin-4α,8-epicatechin has not been isolated from natural sources. In fact no stereoselective synthesis of any procyanidin containing a 4α-linked unit has been reported to date.
It is a plausible assumption that, in the course of the formation of the 4β,8-dimer, the 2-aryl group and the 3-oxygen cooperate in directing the approach of the flavan nucleophile to the presumed carbocationic intermediate towards its β-face. Therefore, accessibility of the 4α-stereoisomer by merely modifying the reaction conditions or attaching protecting groups to one or both of the 3-hydroxyl groups was deemed unlikely. Hence, there is a need for a new synthetic process for the preparation of epicatechins substituted at the 4α position. The instant disclosure is addressed to that unmet need.